1. Summary of the Invention
The present invention relates to a method of use of at least one compound isolated from cherries as cyclooxygenase (COX-1 and COX-2) inhibitors. In particular, the present invention provides a natural cherry composition containing a mixture of anthocyanins, bioflavonoids and phenolics for use as anti-inflammatory agents as a result of inhibition of the cyclooxygenase enzymes.
2. Description of Related Art
Many plant-derived compounds may also impart important positive pharmacological or "nutraceutical/phytoceutical" traits to foods by way of their abilities to serve as antioxidants by maintaining low levels of reactive oxygen intermediates, as anti-inflammatory agents by inhibiting prostaglandin synthesis, or as inhibitors of enzymes involved in cell proliferation. These activities may be important in ameliorating chronic diseases including cancer, arthritis, and cardiovascular disease (Kinsella et al., Food Tech. 85-89 (1993). Thus, with natural products, the dietary supplement/food industry and neutraceutical/phytoceutical companies have the opportunity to employ compounds which can not only enhance food stability as effectively as synthetic antioxidants, but can also offer significant health benefits to the consumer.
Cherries are thought to have beneficial health properties in general. Consumption of cherries was reported to alleviate arthritic pain and gout (Hamel, P. B., et al. Cherokee Plants 28: Herald: Raleigh, N. C. (1975)) although there is no evidence for its active components or mode of action. These beneficial effects may be partially associated with the abundance of anthocyanins, the glycosides of cyanidin.
Prunus Cerasus L. (Rosacease), cv. MONTMORENCY is the major tart cherry commercially grown in the United States. In order to challenge the MONTMORENCY monoculture, a new cultivar, BALATON tart cherry (Ujferbertoi furtos), was introduced into the United States in 1984, and has been tested in Michigan, Utah, and Wisconsin. BALATON produces fruits darker than MONTMORENCY.
Colorants like anthocyanins have been regarded as the index of quality in tart cherries. Most importantly, recent results showed that anthocyanins such as cyanidin-3-glucoside have strong antioxidant activities (Tsuda, T., et al, J. Agric. Food Chem. 42:2407-2410 (1994)).
Early studies have showed that MONTMORENCY cherry contains the anthocyanins cyanidin-3-gentiobioside and cyanidin-3-rutinoside (Li, K. C., et al., J. Am. Chem. Soc. 78:979-980 (1956)). Cyanidin-3-glucosylrutinoside was also found in six out of the seven sour cherry varieties (Harborne, J. B., et al., Phytochemistry 3:453-463 (1964)). Dekazos (Dekazos, E. D., J. Food Sci. 35:237-241 (1970)) reported anthocyanin pigments in MONTMORENCY cherry as peonidin-3-rutinoside peonidin and cyanidin along with cyanidin-3-sophoroside, cyanidin-3-rutinoside and cyanidin-3-glucoside. However, cyanidin-3-glucosylrutinoside as well as cyanidin-3-glucoside, cyanidin-3-sophoroside and cyanidin-3-rutinoside were identified as main pigments in sour cherries. Using HPLC retention values, Chandra et al (Chandra, A., et al., J. Agric. Food Chem. 40:967-969 (1992)) reported that cyanidin-3-sophoroside and cyanidin-3-glucoside were the major and minor anthocyanins, respectively, in Michigan grown MONTMORENCY cherry. Similarly, cyanidin-3-xylosylrutinoside was detected as a minor pigment in MONTMORENCY cherry (Shrikhande, A. J. and F. J. Francis, J. Food Sci. 38:649-651 (1973)).
In the prior art, production of pure anthocyanins (compounds 1-3 of FIG. 1) from BALATON and MONTMORENCY cherry juices was carried out first by adsorbing the pigment on an AMBERLITE XAD-2 (Sigma Chemicals) column (Chandra, A., et al., J. Agric. Food Chem. 41:1062-1065 (1993)). The column was washed with water until the eluant gave a pH of approximately 7.0. The adsorbed pigments along with other phenolics were eluted with MeOH. The resulting crude anthocyanins were fractionated and purified by C-18 MPLC and HPLC, respectively, to afford pure anthocyanins for spectral studies. Purification of 500 mg crude MONTMORENCY anthocyanins from AMBERLITE XAD-2 yielded 60 mg of pure anthocyanins 1-3 compared to 391.43 mg from BALATON. This research indicated that crude anthocyanins from MONTMORENCY obtained from the XAD-2 contained a high percentage of other organic compounds. There was no attempt to use the crude mixture of phenolics and anthocyanins for any purpose. U.S. Pat. Nos., 5,266,685 to Garbutt, 5,665,783 to Katzakian et al and 5,817,354 to Mozaffar describe various adsorbent resins and their use for isolating unrelated products. These patents are only illustrative of the general state of the art in the use of adsorbent resins.
Cyclooxygenase (COX) or prostaglandin endoperoxide H synthase (PGHS-1, PGHS-2 or COX-1/COX-2) enzymes are widely used to measure the anti-inflammatory effects of plant products (Bayer, T., et al., Phytochemistry 28 2373-2378 (1989); and Goda, Y., et al., Chem. Pharm. Bull. 40 2452-2457 (1992)). COX enzyme is the pharmacological target site for the nonsteroidal anti-inflammatory drug discovery (Humes, J. L., et al., Proc. Natl. Acad. Sci. U.S.A. 78 2053-2056 (1981); and Rome, L. H., et al., Proc. Natl. Acad. Sci. U.S.A. 72 4863-4865 (1975)). Two isozymes of cyclooxygenase involved in prostaglandin synthesis are cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), respectively (Hemler, M., et al., J. Biol. Chem. 25 251, 5575-5579 (1976)). It is hypothesized that selective COX-2 inhibitors are mainly responsible for anti-inflammatory activity (Masferrer, J. L., et al., Proc. Natl. Acad. Sci. U.S.A. 91 3228-3232 (1994)). Flavonoids are now being investigated as anti-inflammatory substances as well as their structural features for cyclooxygenase (COX) activity. The 5,7-dihydroxyflavone, galangin with an IC.sub.50 of 5.5 .mu.M, was found to be the most active cyclooxygenase inhibitory flavonoid (Wurm, G., et al., Deutche Apotheker Zeitung 122 2062-2068 (1982)). Flavonoids with an ortho-dihydroxy in ring A or B were stronger inhibitors than those with a free 3-OH group (Wurm, G., et al., Deutche Apotheker Zeitung 122 2062-2068 (1982); and Baumann, J., et al., Prostaglandins 20 627-640 (1980)). The C.sub.2 -C.sub.3 double bond, which determines the coplanarity of the hetero rings appears to be a major determinant of COX activity (Wurm, G., et al., Deutche Apotheker Zeitung 122 2062-2068 (1982)). Certain prenylated flavonoids, such as morusin, were also active, because of their higher lipophilicity (Kimura, Y., et al., Chem. Pharm. Bull. 34 1223-1227 (1986)). Also, unsubstituted flavone is a good COX inhibitor (Mower, R. L., et al., Biochem. Pharmacol. 33 357-364 (1984); and Welton, A. F., et al., Prog. Clin. Biol. Res. 213 231-242 (1986)). Most of the flavanones studied in the past did not show significant COX inhibition, except for the flavanone-3-ol, silibinin (Kalkbrenner, F., et al., Pharmacology 44 1-12 (1992)). However, the structure-activity relationships of isoflavonoids are not reported.
There is a need for natural product derived compositions for use as cyclooxygenase inhibitors and as anti-inflammatory agents.